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UNIQ7615c58721c2f84d-partinfo-00000000-QINU UNIQ7615c58721c2f84d-partinfo-00000001-QINU

••••• IgemBsAs 2013

Objective Asess mRFP production, stability and naked eye discernibility range under inducible conditions. General procedure Different assays were performed using E. coli (DH5α strain) harbouring a plasmid that encodes mRFP under arsenite inducible promoter (ArsRFP culture). mRFP production at different arsenite concentrations Method ArsRFP cultures were grown with different arsenite concentrations ( 0, 50, 200 and 1000ppb). 1ml aliquots were taken after 24 hours and fluorescence was measured. Results mRFP fluorescence increases with higher arsenite concentrations, in a sigmoidal way. mRFP production over time Method A 100 ml ArsRFP culture was grown at 30°C until it reached OD=0.4 (OD 600nm). At this point arsenite was added (1000ppb final concentration) and fluorescence was measured every 30 minutes during 8 hours at 584 nm excitation peak and 608 nm emission peak. Results As shown in the figure below, mRFP production increses over time with arsenite (1000 ppb). However, there is a 3 hours lag after inoculation. mRFP stability over time Method ArsRFP culture was grown overnight at 37ºC in 10 ml LB medium with 2000ppb arsenite concentration. The following day, the culture was centrifuged and the supernatant was discarded in order to remove the arsenite, thus stopping the induction. Afterwars, fresh LB medium was added and the pellet was resuspended. This was done twice and the culture was returned to 37ºC incubation. 1 ml of this culture was taken every 12 hours for the following 4 days. Finally, fluorescence was measured. Results mRFP degradation is shown over time, specially during the first 24 hours. Naked eye discernibility range of mRFP production by arsenite inducible promoter Method ArsRFP cultures were grown with different arsenite concentrations ( 0, 10, 50, 200 and 1000ppb). 1ml aliquots were taken every 12 hours and centrifuged at 10.000rpm for 5 minutes. Pellets pictures were taken in order to compare the range of colour at naked eye. mRFP fluorescence was also measured with a fluorimeter at 484nm for excitation and 608 nm for emission. Results As it can be seen in the pictures below, a difference in the colour production can be clearly distinguished between different arsenite concentrations after 24 hours of induction and between the higher arsenite concentrations only. It can also be observed that over time the production grows and that after 62 hours the difference between 200 and 1000ppb is not clear. Induction over time at different concentrations of arsenite (ppb) After 12 hours of induction: After 24 hours of induction: After 36 hours of induction: After 48 hours of induction: After 50 hours of induction: After 62 hours of induction: Overall conclusions mRFP production responds efficiently under inducible conditions, both over time and different arsenite concentrations. The mRFP stability is acceptable for the aim of our Project. However, the visibility to the naked eye is not sufficient at low arsenite concentrations. We reached to the conclusion that, in order to increase the colour production at lower arsenite concentrations, a signal amplification system has to be added. It could also be added some switch to stop the production and avoid saturation of all the samples, and thus keep the range of colour observable to the naked eye.

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••••• Bielefeld-CeBiTec 2015

We measured the time course of the response of this device to different arsenic concentrations. We observed a reaction approximately five hours after addition of arsenic. The safety limit of 10 µg/L could clearly be distinguished from the negative control and the fluorescence signal increased up to a concentration of 500 µg/L. The signal in the presence of 1000 µg/L was slightly lower than in the presence of 500 µg/L.

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